Universal sanger sequencing from next-gen sequencing amplicons

ABSTRACT

Described herein are methods, compositions and kits directed to amplification of nucleic acids suitable for both next generation sequencing (NGS) and a second round of sequencing as validation, such as Sanger sequencing.

TECHNICAL FIELD

The present technology relates to primers and related methods forproviding separate validation of the sequence of amplicons designed alsofor sequence determination by next-generation sequencing (NGS) methods.

BACKGROUND

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

DNA Sequencing

The chain-termination method (also called Sanger sequencing, after thedeveloper) is the dominant method of sequencing. The classicalchain-termination method requires a single-stranded DNA template, a DNAprimer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs),and modified nucleotides (dideoxyNTPs) that terminate DNA strandelongation. These chain-terminating nucleotides lack a 3′-OH grouprequired for the formation of a phosphodiester bond between twonucleotides, causing DNA polymerase to cease extension of DNA when addNTP is incorporated. The ddNTPs may be radioactively or fluorescentlylabeled for detection in automated sequencing machines. There are anumber of “universal sequencing primers” which are incorporated intoplasmids for convenient generation of sequencing constructs. Suchprimers are generally available for free or at relative low prices.

Modifications of the basic Sanger method, and increased automation, havebeen the foundation for most genomic sequencing.

Next Generation Sequencing

DNA sequencing technologies have advanced exponentially. Most recently,high-throughput sequencing (or next-generation sequencing) technologiesparallelize the sequencing process, producing thousands or millions ofsequences at once. In ultra-high-throughput sequencing as many as500,000 sequencing-by-synthesis operations may be run in parallel.Next-generation sequencing lowers the costs and greatly increases thespeed over the industry standard dye-terminator methods.

Massively Parallel Signature Sequencing (MPSS) was one of the earliernext-generation sequencing technologies. MPSS uses a complex approach ofadapter ligation followed by adapter decoding, reading the sequence inincrements of four nucleotides. This method made it susceptible tosequence-specific bias or loss of specific sequences.

Polony sequencing combined an in vitro paired-tag library with emulsionPCR, an automated microscope, and ligation-based sequencing chemistry tosequence an E. coli genome. The technology was incorporated into theApplied Biosystems SOLiD platform.

454 pyrosequencing amplifies DNA inside water droplets in an oilsolution (emulsion PCR), with each droplet containing a single DNAtemplate attached to a single primer-coated bead that then forms aclonal colony. The sequencing machine contains many picoliter-volumewells each containing a single bead and sequencing enzymes.Pyrosequencing uses luciferase to generate light for detection of theindividual nucleotides added to the nascent DNA, and the combined dataare used to generate sequence read-outs.

In Solexa sequencing DNA molecules and primers are first attached on aslide and amplified with polymerase so that local clonal colonies,initially coined “DNA colonies”, are formed. To determine the sequence,four types of reversible terminator bases (RT-bases) are added andnon-incorporated nucleotides are washed away. Unlike pyrosequencing, theDNA chains are extended one nucleotide at a time and image acquisitioncan be performed at a delayed moment, allowing for large arrays of DNAcolonies to be captured by sequential images taken from a single camera.

SOLiD technology employs sequencing by ligation. Here, a pool of allpossible oligonucleotides of a fixed length are labeled according to thesequenced position. Oligonucleotides are annealed and ligated; thepreferential ligation by DNA ligase for matching sequences results in asignal informative of the nucleotide at that position. Beforesequencing, the DNA is amplified by emulsion PCR. The resulting beads,each containing single copies of the same DNA molecule, are deposited ona glass slide. The result is sequences of quantities and lengthscomparable to Solexa sequencing.

Validation

While next-generation sequence methods are faster and cheaper thantraditional methods, there remain questions as to their accuracy.Accuracy is particularly important in human clinical diagnostics, wheresignificant medical decisions may hinge on a single nucleotidepolymorphism.

In view of the higher fidelity requirements for diagnostic applications,recent clinical guidelines have mandated that all mutations identifiedby next-generation sequencing must be validated on an alternativeplatform before reporting the results, such as the Sanger method. See,e.g. www.osehra.org/blog/big-news-next-generation-sequencing-guidelines-issued-college-american-pathologists,Aug. 1, 2012; see also, American College of Medical Genetics ACMGStandards and Guidelines for Clinical Laboratories.

SUMMARY OF THE INVENTION

Described herein are methods, compositions, and kits for theamplification and sequencing of nucleic acids, particularly inconjunction with next-generation sequencing.

In one embodiment, the present disclosure provides a compositioncomprising a first oligonucleotide that comprises, in 5′ to 3′ order,(a) a first region suitable for use as a sequencing primer (e.g., aSanger sequencing primer); (b) a spacer that is between about 5 andabout 15 nucleotides long; and (c) a third region suitable for use as asequencing primer (e.g., a next generation sequencing (NGS) primer), anda second oligonucleotide that comprises, in 5′ to 3′ order, (d) a firstregion that is substantially identical to the third region of the firstoligonucleotide; and (e) a second region that is suitable for use as apolymerase chain reaction (PCR) primer, wherein the third region of thefirst oligonucleotide and the first region of the second oligonucleotidehave a melting temperature (Tm) that is at least about 5° C. higher thanthe Tm of the second region of the second oligonucleotide, and whereinthe total length of the spacer and the first and second regions of thesecond oligonucleotide is at least about 45 nucleotides (nt) long.

In some aspects, the first oligonucleotide is not longer than about 80nt, 90 nt, 100 nt, 110 nt, or 120 nt. In some aspects, the secondoligonucleotide is not longer than about 50 nt, 60 nt, 70 nt or 80 nt.In some aspects, the Tm of the third region of the first oligonucleotideand the first region of the second oligonucleotide is between about 65°C. and about 75° C. In some aspects, the Tm of the second region of thesecond oligonucleotide is between about 55° C. and about 65° C.

In some aspects, the first region of the first oligonucleotide isselected from Table 1. In some aspects, the third region of the firstoligonucleotide and the first region of the second oligonucleotide areselected from Table 2.

In some aspects, the composition further comprises a thirdoligonucleotide that comprises, in 5′ to 3′ order, (a) a first regionsuitable for use as a sequencing primer (e.g., a Sanger sequencingprimer); (b) a spacer that is between about 5 and about 15 nucleotideslong; and (c) a third region suitable for use as a sequencing primer(e.g., a next generation sequencing (NGS) primer), and a fourtholigonucleotide that comprises, in 5′ to 3′ order, (d) a first regionthat is substantially identical to the third region of the thirdoligonucleotide; and (e) a second region that is suitable for use as apolymerase chain reaction (PCR) primer, wherein the third region of thethird oligonucleotide and the first region of the fourth oligonucleotidehave a melting temperature (Tm) that is at least about 5° C. higher thanthe Tm of the second region of the fourth oligonucleotide, wherein thetotal length of the spacer of the third oligonucleotide and the firstand second regions of the fourth oligonucleotide is at least about 45nucleotides (nt) long, and wherein the first region of the firstoligonucleotide is substantially different from the first region of thethird oligonucleotide and the first region of the second oligonucleotideis substantially different from the first region of the fourtholigonucleotide.

In some aspects, the second region of the second oligonucleotide and thesecond region of the fourth oligonucleotide are suitable for amplifyinga human genomic sequence.

Also provided, in one embodiment, is an oligonucleotide comprising, in5′ to 3′ order: (a) a first region selected from the nucleic acidsequences of Table 1; (b) a spacer that is between about 5 and about 15nucleotides long; and (c) a third region selected from the nucleic acidsequences of Table 2. In some aspects, the oligonucleotide comprises asequence of

-   GTAAAACGACGGCCAGTATTTAGGTGACACTATAGACACTGACGACATGGTTCT ACA (SEQ ID    NO: 9) or-   AACAGCTATGACCATGCAGTCAAGTAACAACCGCGATACGGTAGCAGAGACTTG GTCT (SEQ ID    NO: 10).

In another embodiment, provided is a method for amplifying a nucleotidesequence, comprising: (i) incubating a target nucleotide template with afirst and third oligonucleotides each comprising, in 5′ to 3′ order, (a)a first region suitable for use as a sequencing primer (e.g., a Sangersequencing primer); (b) a spacer that is between about 5 and about 15nucleotides long; and (c) a third region suitable for use as asequencing primer (e.g., a next generation sequencing (NGS) primer), anda second and fourth oligonucleotides comprising, in 5′ to 3′ order, (d)first regions that are substantially identical to the third region ofthe first or third oligonucleotide, respectively; and (e) second regionswhich, in combination, suitable for amplifying the target nucleotidetemplate as a pair of polymerase chain reaction (PCR) primers, whereinthe third regions of the first and third oligonucleotides and the firstregions of the second and fourth oligonucleotides have a meltingtemperature (Tm) that is at least about 5° C. higher than the Tm of thesecond regions of the second and fourth oligonucleotide, and wherein thetotal length of the spacer of the first oligonucleotide and the firstand second regions of the second oligonucleotide is at least about 45nucleotides (nt) long and the total length of the spacer of the thirdoligonucleotide and the first and second regions of the fourtholigonucleotide is at least about 45 nucleotides (nt) long, and whereinthe first region of the first oligonucleotide is substantially differentfrom the first region of the third oligonucleotide and the first regionof the second oligonucleotide is substantially different from the firstregion of the fourth oligonucleotide; (ii) performing a plurality of PCRcycles with a first annealing temperature (Ta) suitable foramplification using the second regions of the second and fourtholigonucleotides as primers; and (iii) performing a plurality of PCRcycles with a second annealing temperature (Ta) suitable foramplification using the third regions of the first and thirdoligonucleotides as primers.

In some aspects, the first regions of the first and thirdoligonucleotides are selected from Table 1. In some aspects, the firstregions of the third and fourth oligonucleotides are selected from Table2.

In some aspects, the first Ta is between about 53° C. and about 57° C.In some aspects, the second Ta is between about 59° C. and about 63° C.

In some aspects, the ratios of concentrations of the firstoligonucleotide to the second oligonucleotide and the thirdoligonucleotide to the fourth oligonucleotide are less than about 1:1.

Also provided, in one embodiment, is a composition or kit, comprising afirst and second oligonucleotides each comprising, in 5′ to 3′ order,(a) a first region suitable for use as a sequencing primer (e.g., aSanger sequencing primer); (b) a spacer that is between about 5 andabout 15 nucleotides long; and (c) a third region suitable for use as asequencing primer (e.g., a next generation sequencing (NGS) primer),wherein the first regions of the first and second oligonucleotides aresubstantially different and the third regions of the first and secondoligonucleotides are substantially different.

In some aspects, the first regions of the first and secondoligonucleotides are selected from Table 1. In some aspects, the thirdregions of the first and second oligonucleotides are selected from Table2.

With reference to FIGS. 1 and 2, one embodiment of the presentdisclosure provides an amplification method that entails PCRamplification with two pairs of primers.

An inner primer set is used for generating an amplicon for a first roundof sequencing, such as a Next Generation Sequencing (NGS). Each of theinner primers includes, from 5′ to 3′, a first region suitable for useas primer for sequencing and a second region that is specific for atarget nucleotide sequence. The sequencing primer regions are referredto as Adaptors A (for forward sequencing) and B (for reversesequencing), as shown in FIG. 2. Also as shown in FIG. 2, the secondregions are referred to as gene-specific primers forward (GSP F) andreverse (GSP R).

Such inner primer sets are similar to those used in NGS, in which theGSPs serve as primers for the target-specific PCR amplifications, andthe Adaptors are then used for sequencing the amplicons.

Unlike the inner primer set, the outer primer set does not include anysequence that is specific to the target nucleotide sequence. At the 3′end, each of the outer primers includes a region (third region) that isidentical or substantially identical to Adaptor A or B. Then, at the 5′end, each of the outer primers includes a first region that is suitablefor initiation a second round of sequencing (e.g., Sanger sequencing).Accordingly, these first regions can be referred to as “Sanger primerregions,” without being limited to primers useful for Sanger sequencing.

A nucleic acid sequence being “substantially identical” to a referencenucleic acid, as used herein, means that the nucleic acid is able tohybridize, under suitable or designated hybridizing conditions, to thecomplementary strand of the reference nucleic acid. In some aspects, thenucleic has at most 1, or 2 or 3 nucleotide mismatches ordeletion/insertion from the reference nucleic acid.

Therefore, a first cycle of amplification with the inner primer set willgenerate an amplicon that includes, at both ends, the inner primersequences. Such an amplicon, therefore, can subsequently undergo PCRamplification with the outer primer set as well, by virtue of thesequence identity between the 3′ ends of the outer primers and the 5′ends of the inner primers. Which primer set is used in the subsequentPCR cycles can depend on the availability of the primers as well asfactors such as annealing temperature.

At the end of the PCR amplification, therefore, at least two types ofamplicons are generated. One of them includes both inner primers at theends, and the other includes both outer primers at the ends. The laterone is suitable for sequencing with either the Sanger primers regions orthe Adaptor regions. Accordingly, the above amplification methods cangenerate amplicons suitable for two separate rounds and types ofsequencing. One exemplary use of such an amplicon is for a NGSsequencing followed by a Sanger sequencing validation.

It is further discovered that the inner and outer primer sets can bedesigned to favor PCR amplification with either set of the primers whileneeded. For instance, in one embodiment, the Adaptors can be nucleicacid sequences that have a relatively high melting temperature (T_(m))and the target-specific primers can have a relatively lower T_(m). Assuch, when the annealing temperature (T_(a)) is low, the amplificationpreferentially utilizes the inner primers. By the same token, when theT_(a) is higher, the amplification preferentially utilizes the outerprimers.

For instance, if the Adaptors' T_(m) is around 70° C. and the GSPs'T_(m) is around 60° C., a T_(a) of about 55° C. will strongly favor theGSPs and a T_(a) of about 62° C. will strongly favor the Adaptors. Inaccordance with such a design, the present disclosure provides a PCRreaction in which the amplification is carried out, for a number ofcycles, with a lower T_(a), to ensure that a sufficient number ofamplicons is generated, having the inner primers at both ends.Subsequently, cycles with increased T_(a) will enable such amplicons toundergo amplification with the outer primers.

In some aspects, the Adaptors have a T_(m) that is at least about 5° C.,or 6° C., 7° C., 8 ° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C.,or 15° C. higher than the T_(m) of the GSPs. In some aspects, the PCRamplifications are carried out first in a number of cycles with a T_(a)between about 53° C. and about 57° C., or between about 54° C. and about56° C., or at about 55° C. Subsequently, the PCR amplifications arecarried out in a number of cycles with a T_(a) between about 59° C. andabout 65° C., or between about 60° C. and about 64° C., or between about61° C. and about 63° C., or at about 62° C.

In some aspects, the PCR is carried out under the lower T_(a) for atleast 10 cycles. In some aspects, the PCR is then carried out under thehigher T_(a) for at least about 20 cycles.

In some aspects, the outer primers further include a spacer regionbetween the Sanger primer region and the Adaptors. In one aspect, thetotal length of the spacer, the Adaptor and the GSP is at least about 50nucleotides such that the Sanger sequencing can effectively begin at thetarget sequence right next to the GSP. This is because Sanger sequencinggenerally starts at about 50 nt from the primer site. In some aspects,the total length is at least about 55 nt, 60 nt, or 65 nt. In someaspects, the spacer is between about 5 nt and about 15 nt long. In oneaspect, the spacer is at least about 6, or 7, 8, 9, 10, 11, or 12 ntlong.

The present technology, therefore, is able to provide primer setssuitable for amplify multiple target nucleic acid sequences (usingtarget or gene-specific primers), using the present methods, with theresulting amplicons suitable for two types of sequencing. In thisrespect, it is noted that methods and packages/kits are also provided,employing multiple oligonucleotide sets as described here. Thesemultiple oligonucleotide sets each includes different PCR primerstargeting different nucleic acid sequences or genes of interest, butincludes the same Sanger and NGS sequencing primers. In some aspects,the methods or packages/kits include at least 2, or 3, or 4, or 5, or10, or 20, or 30, or 40 or 50, or 100 such oligonucleotide sets.

Methods are known in the art to design primer sequences for sequencing,such as Sanger sequencing. In one aspect, the Sanger sequencing primerregion is a sequence selected from Table 1. In another aspect, theAdaptor region is a sequence selected from Table 2.

TABLE 1 Exemplary Universal Primers for Sanger Sequencing Size SEQPrimer Sequence(5′ -> 3′) (nt) ID NO. M13F GTAAAACGACGGCCAGT 17  1 M13RAACAGCTATGACCATG 16  2 M13-26REV CAGGAAACAGCTATGAC 17 11 M13-40FORGTTTTCCCAGTCACGAC 17 12 M13-48REV CGGATAACAATTTCACACAG 20 13 Ml3F-20GTAAAACGACGGCCAGTG 18 14 1629DWN CTGTAAATCAACAACGCACAG 21 15 3AOX1<GCAAATGGCATTCTGACATCC 21 16 5A0X1< GACTGGTTCCAATTGACAAGC 21 17 A-FACTORTACTATTGCCAGCATTGCTGC 21 18 ACYCDUETUP1 GGATCTCGACGCTCTCCCT 19 19 ATTB1GTTTGTACAAAAAAGCAGGC 20 20 ATTB2 CCACTTTGTACAAGAAAGCTGGGT 24 21 ATTL1CGCGTTAACGCTAGCATGGATCTC 24 22 ATTL2 CATCAGAGATTTTGAGACAC 20 23 BD-FORTCATCGGAAGAGAGTAG 17 24 BD-REV CGTTTTAAAACCTAAGAGTCA 21 25 BGHR2GGGTCAAGGAAGGCACG 17 26 BGHREV TAGAAGGCACAGTCGAGG 18 27 CMV-FORCGCAAATGGGCGGTAGGCGTG 21 28 DUETDOWN1 GATTATGCGGCCGTGTACAA 20 29EF-1-ALPHA TCAAGCCTCAGACAGTGGTTC 21 30 EGFP-C-FOR CATGGTCCTGCTGGAGTTCGTG22 31 EGFP-C-REV GTTCAGGGGGAGGTGTG 17 32 EGFP-N CGTCGCCGTCCAGCTCGACCAG22 33 GAL1- ATTTTCGGTTTGTATTACTTC 21 34 FORWARD GAL4AD TACCACTACAATGGATG17 35 GL-PRIMER1 TGTATCTTATGGTACTGTAACTG 23 36 GL-PRIMER2CTTTATGTTTTTGGCGTCTTCCA 23 37 ITS1 TCCGTAGGTGAACCTGCGG 19 38 ITS4TCCTCCGCTTATTGATATGC 20 39 ITSL TCGTAACAAGGTTTCCGTAGGTG 23 40 KSTCGAGGTCGACGGTATC 17 41 MAL-E GGTCGTCAGACTGTCGATGAAGCC 24 42 OP1E2FORCGCAACGATCTGGTAAACAC 20 43 OP1E2REV GACAATACAAACTAAGATTTAGCT 24 44 PBADFATGCCATAGCATTTTTATCC 20 45 PBADR GATTTAATCTGTATCAGG 18 46 PCEP-FORAGAGCTCGTTTAGTGAACCG 20 47 PCEP-REV GTGGTTTGTCCAAACTCATC 20 48 PDON-RGTAACATCAGAGATTTTGAGACAC 24 49 PENTR-1A- GTTTCTACAAACTCTTCCTG 20 50 FORPET- ATGCGTCCGGCGTAGA 16 51 UPSTREAM PETBLUEDOWN GTTAAATTGCTAACGCAGTCA21 52 PETBLUEUP TCACGACGTTGTAAAACGAC 20 53 PFASTBAC-F CATACCGTCCCACCATCG18 54 PFASTBAC-R ATCCTCTAGTACTTCTCGAC 20 55 PGEX3PCCGGGAGCTGCATGTGTCAGAGG 23 56 PGEX5FUPSTM TGGACCCAATGTGCCTG 17 57 PGEX5PGGGCTGGCAAGCCACGTTTGGTG 23 58 PINDIGO-REV CTCGTATGTTGTGTGGAATTGTGAGC 2659 PIRES-REV CATATAGACAAACGCACAC 19 60 PJG4-5-FORGATGCCTCCTACCCTTATGATGTGCC 26 61 POLYTV TTTTTTTTTTTTTTTTTTTTTTTV 24 62PQE-FOR CCCGAAAAGTGCCACCTG 18 63 PQE-REV GTTCTGAGGTCATTACTG 18 64PREP-FOR GCTCGATACAATAAACGCC 19 65 PSHUTCMV- GGTCTATATAAGCAGAGCTG 20 66FOR PSHUTCMV- GTGGTATGGCTGATTATGATCAG 23 67 REV PTRC-99A-GACATCATAACGGTTCTG 18 68 FOR PTRC-99A- CTGAGTTCGGCATGGGG 17 69 REVPTRCHIS-F GAGGTATATATTAATGTATCG 21 70 PTRCHIS-R GATTTAATCTGTATCAGG 18 71RVPRIMER3 CTAGCAAAATAGGCTGTCCC 20 72 RVPRIMER4 GACGATAGTCATGCCCCGCG 2073 S.TAG CGAACGCCAGCACATGGACA 20 74 SK-PRIMER CGCTCTAGAACTAGTGGATC 20 75SP6 ATTTAGGTGACACTATAG 18 76 T3P ATTAACCCTCACTAAAGGGA 20 77 T7-REVTAGTTATTGCTCAGCGGTGG 20 78 T7- AACCCCTCAAGACCCGTTTA 20 79 SELECTDWN T7PTAATACGACTCACTATAGGG 20 80 T7TERM CTAGTTATTGCTCAGCGG 18 81 TRX-FORWARDTTCCTCGACGCTAACCTG 18 82 U-19 GTTTTCCCAGTCACGACGT 19 83

TABLE 2 Exemplary Next Generation Sequencing Primers Size SEQ PrimerSequence(5′ -> 3′) (nt) ID NO. Adaptor A ACACTGACGACATGGTTCTACA 22 7Adaptor B TACGGTAGCAGAGACTTGGTCT 22 8

In some embodiments, the inner and outer primer sets are present inrelative ratios of greater than about 1:1. In one embodiment, the innerand out primer sets have a ratio that is at about 2.5:1, or betweenabout 2:1 and 3:1, or between about 1.5:1 and 4:1, or between about 1:1and 5:1.

In one embodiment, an outer primer is provided that comprises, in 5′ to3′ order (a) a 5′ sequence derived from M13 sequencing primers, whereinthe sequence comprises GTAAAACGACGGCCAGT (SEQ ID NO: 1) orAACAGCTATGACCATG (SEQ ID NO: 2); (b) a spacer comprising at least 10nucleotides, and (c) a 3′ adapter that is substantially identical to the5′ region of primers used in the next generation sequencingamplification. In related embodiments, the spacer comprises a sequenceselected from ATTTAGGTGACACTATAG (SEQ ID NO: 3) or CAGTCAAGTAACAACCGCGA(SEQ ID NO: 4). For example, the oligonucleotide primer may comprise asequence selected from GTAAAACGACGGCCAGTATTTAGGTGACAC TATAG (SEQ ID NO:5) or AACAGCTATGACCATGCAGTCAAGTAACAACCGCGA (SEQ ID NO: 6). In furtherembodiments, the adapter comprises a sequence selected from ACACTGACGACATGGTTCTACA (SEQ ID NO: 7) or TACGGTAGCAGAGACTT GGTCT (SEQ ID NO: 8).Thus, in an exemplary embodiment, the oligonucleotide primer comprises asequence selected from GTAAAACGACGGCCAGTATTTAGGTGACACTA TAGACACTGACGACATGGTTCTACA (SEQ ID NO: 9) or AACAGCTATGACC ATGCAGTCAAGTAACAACCGCGATACGGTAGCAGAGACTTGGTCT (SEQ ID NO: 10).

In one embodiment, the invention is a method for generating an ampliconfor next generation sequencing with subsequent validation, for at leastone region of interest, comprising (a) adding, to a sample of DNA, (i) afirst primer set specific for the region of interest, consisting of twoprimers each comprising a 5′ region encoding an adapter and a 3′ regionspecific for the region of interest, wherein the first primer set bindswith a T_(m) of approximately 60° C. immediately upstream and downstreamof the region of interest;

-   (ii) a second primer set consisting of two primers each of which    comprises

(A) a 5′ sequence selected from GTAAAACGACGGCCAGT (SEQ ID NO: 1) orAACAGCTATGACCATG (SEQ ID NO: 2);

(B) a spacer comprising at least 10 nucleotides

(C) a 3′ adapter that is substantially identical to the adapter region,wherein the adapter has a T_(m) of approximately 55° C., and wherein thesecond primer set has a Tm of at least 70° C.; followed by thermocyclingfor about 10 cycles with 55° C. annealing and about 30 cycles at 60-62°C. annealing. For example, thermocycling may comprise (a) heating to 95°C.; (b) extending for about 10 cycles of (i) 95° C. (ii) 55° C. (ii) 72°C.; and (c) extending for about 30 cycles of (i) 95° C. (ii) 60-62° C.(ii) 72° C. In some embodiments, an additional extension step is added,such as 72° C. for 15 minutes. In such methods, the ratio of the firstprimer set and the second primer set may affect the efficiency andspecificity of the amplification.

The amplicons generated may then be subject to sequencing, such as bythe addition of a primer selected from GTAAAACGACGGCCAGT (SEQ ID NO: 1)and AACAGCTATGACCATG (SEQ ID NO: 2); extending in the presence of dNTPsand dye-labelled dNTPs to generate a population of labeled nucleotides;and resolving the population of labeled nucleotides.

The invention also includes compositions and kits. In one aspect, thecomposition or kit includes a forward inner primer and a forward outerprimer as described. In one aspect, the composition or kit includes aforward outer primer and a reverse outer primer as described. In anotheraspect, the composition or kit includes an inner primer set and an outerprimer set as described.

In one embodiment, the invention is a kit for the amplification andsequencing of a region of interest, comprising (i) a first primer setspecific for the region of interest, consisting of two primers eachcomprising a 5′ region encoding an adapter and a 3′ region specific forthe region of interest, wherein the first primer set binds with a Tm ofapproximately 60° C. immediately upstream and downstream of the regionof interest; (ii) a second primer set consisting of two primers each ofwhich comprises (A) a 5′ sequence selected from GTAAAACGACGGCCAGT (SEQID NO: 1) or AACAGCTATGACCATG (SEQ ID NO: 2);(B) a spacer comprising atleast 10 nucleotides (C) a 3′ adapter that is substantially identical tothe adapter region, wherein the adapter has a Tm of approximately 55°C., and wherein the second primer set has a Tm of at least 70° C. In anexemplary embodiment, the second primer set comprises:GTAAAACGACGGCCAGTATTTAGGTGACACTATAGACACTGACGACATGGTTCT ACA (SEQ ID NO:9); and AACAGCTATGACCATGCAGTCAAGTAACAACCGCGATA CGGTAGCAGAGACTTGGTCT (SEQID NO: 10). Kits of the invention may further comprise suitable buffersand controls.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Simultaneous nested PCR allows addition of Sanger sequencingpriming sites and extension linkers by universal priming of adaptersequences.

FIG. 2. Thermocycling parameters favoring generation of full lengthamplicon.

FIG. 3A-3C. Results from varying thermocycling and primer ratioparameters. P, PIK3CA-specific inner primers; N, NGS universal outerprimers.

FIG. 4. Comparison of AccessArray thermocycling conditions to the Questmodified conditions favoring full length amplicon production(thermocycling condition #3). All reaction conditions were identicalwith only the thermocycling parameters changed.

FIG. 5A-5B. Sequencing results from purified full length PIK3CA_(—)3product. Forward sequence (A), and reverse sequence (B) both containclean, interpretable sequence (SEQ ID NO: 11) covering the region ofinterest (blue bar).

DETAILED DESCRIPTION

Described herein are primers, methods, reagents and kits forindependently validating the DNA sequence of an amplicon that was, orwill be, subjected to next-generation sequencing.

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” also include the plural. Thus, for example, a reference to “anoligonucleotide” includes a plurality of oligonucleotide molecules, areference to label is a reference to one or more labels, a reference toprobe is a reference to one or more probes, and a reference to “anucleic acid” is a reference to one or more polynucleotides.

As used herein, unless indicated otherwise, when referring to anumerical value, the term “about” means plus or minus 10% of theenumerated value.

The terms “amplification” or “amplify” as used herein includes methodsfor copying a target nucleic acid, thereby increasing the number ofcopies of a selected nucleic acid sequence. Amplification may beexponential or linear. A target nucleic acid may be either DNA or RNA.The sequences amplified in this manner form an “amplification product,”also known as an “amplicon.” While the exemplary methods describedhereinafter relate to amplification using the polymerase chain reaction(PCR), numerous other methods are known in the art for amplification ofnucleic acids (e.g., isothermal methods, rolling circle methods, etc.).The skilled artisan will understand that these other methods may be usedeither in place of, or together with, PCR methods. See, e.g., Saiki,“Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds.,Academic Press, San Diego, Calif. 1990, pp. 13-20; Wharam et al.,Nucleic Acids Res., 29(11):E54-E54, 2001; Hafner et al., Biotechniques,30(4):852-56, 858, 860, 2001; Zhong et al., Biotechniques, 30(4):852-6,858, 860, 2001.

A key feature of PCR is “thermocycling” which, in the present context,comprises repeated cycling through at least three differenttemperatures: (1) melting/denaturation, typically at 95° C. (2)annealing of a primer to the target DNA at a temperature determined bythe melting point (Tm) of the region of homology between the primer andthe target and (3) extension at a temperature dependent on thepolymerase, most commonly 72° C. These three temperatures are thenrepeated numerous times. Thermocycling protocols typically also includea first period of extended denaturation, and end on an extended periodof extension.

The Tm of a primer varies according to the length, G+C content, and thebuffer conditions, among other factors. As used herein, Tm refers tothat in the buffer used for the reaction of interest.

As used herein, the term “detecting” refers to observing a signal from adetectable label to indicate the presence of a target. Morespecifically, detecting is used in the context of detecting a specificsequence.

The terms “complement,” “complementary” or “complementarity” as usedherein with reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a genomic nucleic acid)related by the base-pairing rules. The complement of a nucleic acidsequence as used herein refers to an oligonucleotide which, when alignedwith the nucleic acid sequence such that the 5′ end of one sequence ispaired with the 3′ end of the other, is in “antiparallel association.”For example, for the sequence 5′-A-G-T-3′ is complementary to thesequence 3′-T-C-A-5′. Certain bases not commonly found in naturalnucleic acids may be included in the nucleic acids of the presentinvention and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.Complementarity may be “partial” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete,” “total,” or “full” complementarity between thenucleic acids.

The term “detectable label” as used herein refers to a molecule or acompound or a group of molecules or a group of compounds associated witha probe and is used to identify the probe hybridized to a genomicnucleic acid or reference nucleic acid.

A “fragment” in the context of a gene fragment or a chromosome fragmentrefers to a sequence of nucleotide residues which are at least about 10nucleotides, at least about 20 nucleotides, at least about 25nucleotides, at least about 30 nucleotides, at least about 40nucleotides, at least about 50 nucleotides, at least about 100nucleotides, at least about 250 nucleotides, at least about 500nucleotides, at least about 1,000 nucleotides, at least about 2,000nucleotides.

The terms “identity” and “identical” refer to a degree of identitybetween sequences. There may be partial identity or complete identity. Apartially identical sequence is one that is less than 100% identical toanother sequence. Partially identical sequences may have an overallidentity of at least 70% or at least 75%, at least 80% or at least 85%,or at least 90% or at least 95%.

As used herein, the terms “isolated,” “purified” or “substantiallypurified” refer to molecules, such as nucleic acid, that are removedfrom their natural environment, isolated or separated, and are at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated. An isolatedmolecule is therefore a substantially purified molecule.

The term “multiplex PCR” as used herein refers to an assay that providesfor simultaneous amplification and detection of two or more productswithin the same reaction vessel. Each product is primed using a distinctprimer pair. A multiplex reaction may further include specific probesfor each product that are detectably labeled with different detectablemoieties.

The term “Nested polymerase chain reaction” is a modification ofpolymerase chain reaction which, in the present context, is performed toadd sequences to an amplicon. Nested polymerase chain reaction involvestwo sets of primers, used in two successive runs of polymerase chainreaction, the second set intended to amplify the target from the firstrun product.

As used herein, the term “oligonucleotide” refers to a short polymercomposed of deoxyribonucleotides, ribonucleotides, or any combinationthereof Oligonucleotides are generally between about 10, 11, 12, 13, 14,15, 20, 25, or 30 to about 150 nucleotides (nt) in length, morepreferably about 10, 11, 12, 13, 14, 15, 20, 25, or 30 to about 70 nt.

As used herein, a “primer” is an oligonucleotide that is complementaryto a target nucleotide sequence and leads to addition of nucleotides tothe 3′ end of the primer in the presence of a DNA or RNA polymerase. The3′ nucleotide of the primer should generally be identical to the targetsequence at a corresponding nucleotide position for optimal extensionand/or amplification. The term “primer” includes all forms of primersthat may be synthesized including peptide nucleic acid primers, lockednucleic acid primers, phosphorothioate modified primers, labeledprimers, and the like. As used herein, a “forward primer” is a primerthat is complementary to the anti-sense strand of DNA. A “reverseprimer” is complementary to the sense-strand of DNA.

An oligonucleotide (e.g., a probe or a primer) that is specific for atarget nucleic acid will “hybridize” to the target nucleic acid undersuitable conditions. As used herein, “hybridization” or “hybridizing”refers to the process by which an oligonucleotide single strand annealswith a complementary strand through base pairing under definedhybridization conditions. It is a specific, i.e., non-random,interaction between two complementary polynucleotides. Hybridization andthe strength of hybridization (i.e., the strength of the associationbetween the nucleic acids) is influenced by such factors as the degreeof complementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid.

The term “adapter” refers to a short, chemically synthesized, DNAmolecule which is used to link the ends of two other DNA molecules, orto provide a common template for other manipulations, such assequencing. In the present context, an adapter is used innext-generation sequencing as the basis for sequencing.

“Specific hybridization” is an indication that two nucleic acidsequences share a high degree of complementarity. Specific hybridizationcomplexes form under permissive annealing conditions and remainhybridized after any subsequent washing steps. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may occur, for example, at 65° C. in thepresence of about 6×SSC. Stringency of hybridization may be expressed,in part, with reference to the temperature under which the wash stepsare carried out. Such temperatures are typically selected to be about 5°C. to 20° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Equations forcalculating T_(m) and conditions for nucleic acid hybridization areknown in the art.

As used herein, an oligonucleotide is “specific” for a nucleic if it iscapable of hybridizing to the target of interest and not substantiallyhybridizing to nucleic acids which are not of interest. High levels ofsequence identity are preferred and include at least 75%, at least 80%,at least 85%, at least 90%, at least 95% and more preferably at least98% sequence identity. Sequence identity can be determined using acommercially available computer program with a default setting thatemploys algorithms well known in the art (e.g., BLAST).

The term “region of interest” refers to a region of a nucleic acid to besequenced.

The term “transcript,” when referring to a target nucleic acid, refersto any nucleic acid transcript, including mRNA, pre-mRNA, and snRNA, andsynthetic representations thereof such as cDNA.

The term “biological sample” as used herein refers to a samplecontaining nucleic acids of interest. A biological sample may compriseclinical samples (i.e., obtained directly from a patient) or isolatednucleic acids and may be cellular or acellular fluids and/or tissue(e.g., biopsy) samples. In some embodiments, a sample is obtained from atissue or bodily fluid collected from a subject. Sample sources include,but are not limited to, sputum (processed or unprocessed), bronchialalveolar lavage (BAL), bronchial wash (BW), whole blood or isolatedblood cells of any type (e.g., lymphocytes), bodily fluids,cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsymaterial). Methods of obtaining test samples and reference samples arewell known to those of skill in the art and include, but are not limitedto, aspirations, tissue sections, drawing of blood or other fluids,surgical or needle biopsies, collection of paraffin embedded tissue,collection of body fluids, collection of stool, and the like. In thepresent context the biological sample preferably is blood, serum orplasma. The term “patient sample” as used herein refers to a sampleobtained from a human seeking diagnosis and/or treatment of a disease,especially prostate disease.

As used herein, the term “subject” refers to a mammal, such as a human,but can also be another animal such as a domestic animal (e.g., a dog,cat, or the like), a farm animal (e.g., a cow, a sheep, a pig, a horse,or the like) or a laboratory animal (e.g., a monkey, a rat, a mouse, arabbit, a guinea pig, or the like). The term “patient” refers to a“subject” who possesses, or is suspected to possess, a geneticpolymorphism of interest.

Amplification of Nucleic Acids. Nucleic acid samples or target nucleicacids may be amplified by various methods known to the skilled artisan.In suitable embodiments, PCR is used to amplify nucleic acids ofinterest. Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleotide triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase.

In one embodiment, the target nucleic acids are amplified in a multiplexamplification or nested reaction. If the target sequence is present in asample, the primers will bind to the sequence and the polymerase willcause the primers to be extended along the target sequence by adding onnucleotides. By raising and lowering the temperature of the reactionmixture, the extended primers will dissociate from the target nucleicacid to form reaction products, excess primers will bind to the targetnucleic acid and to the reaction products and the process is repeated,thereby generating amplification products. Cycling parameters can bevaried, depending on the length of the amplification products to beextended. An internal positive amplification control (IC) can beincluded in the sample, utilizing oligonucleotide primers and/or probes.

Detection of Amplified Nucleic Acids. Amplification of nucleic acids canbe detected by methods known in the art such as gel electrophoresis,column chromatography, hybridization with a probe, sequencing, meltingcurve analysis, or “real-time” detection.

General Overview of the Technology. Next generation sequencing methodsare fast and low cost compared to older technologies. However, in aclinical diagnostic setting, accuracy is extremely important. Therefore,following a next-generation sequencing protocol, such as Ion Torrent,any clinically relevant results need to be confirmed and validated by anindependent and more reliable technique. Thus, when a region of interestis amplified and sequenced with the aid of a set of first primers, thereis required a way to verify the sequencing results.

Applicants have met this need by providing a second set of primers thatextend an amplicon of interest to add well understood and characterized“universal” primers and a spacer region. The resulting amplicon can thenbe sequenced with a universal primer. The spacer region ensures thathighly accurate sequencing, which typically starts 50 nucleotides fromthe primer, encompasses the region of interest.

The amplification of a region of interest with the second set of primerscan be done in a separate reaction. For example, the region of interestcan be amplified with the first set of primers and sequences with nextgeneration techniques. After identifying an amplicon of interest, theamplicon can then be further amplified and tagged with the second set ofprimers, and resequenced.

Preferably, however, amplification with the first and second set ofprimers occurs using nested PCR to result in an amplicon that is firstsequenced with next generation technology and then with oldertechnology. This approach avoids errors from repeated amplification.

EXAMPLES Example 1 Proof of Concept with PIK3CA Primers

We developed a primer set for Universal Sanger Sequencing from Next GenSequencing amplicons.

This design takes advantage of the universal adapters that areincorporated into each amplicon of a Next Gen Sequencing library. Forexample, Ion Torrent PGM incorporates the forward (adapter A) andreverse (adapter B) shown in Table 3. Primers for each amplicon makingup the library are tagged with adapter A on the 5′ region of the forwardprimer and adapter B on the 5′ region of the reverse primer.

TABLE 3 Ion Torrent PGM adapters Adapter 5′ --> 3′ Sequence Adapter AACACTGACGACATGGTTCTACA (SEQ ID NO: 7) Adapter B TACGGTAGCAGAGACTTGGTCT(SEQ ID NO: 8)

The universal Sanger sequencing primer set (Table 4) utilizes theseadapter sequences as the priming sites and includes an extension linkeras well as M13F and M13R for sequencing (FIG. 1). The M13 sequencingsites and extension linkers were synthesized onto the product during asimultaneous nested PCR strategy. Briefly, the gene specific primers andUniversal Sanger sequencing primers were added to the same reaction toobtain full length amplicon. The purpose of the extension linkers was toserve as a space buffer to allow clean sequence reads, which generallybegins ˜50 bp from the priming site, to begin near the start of theregion of interest.

TABLE 4 Universal Sanger Sequencing Primers NG2SANG-F: M13F-SP6-AdA(M13F = 17; Entire oligo = 57) (SEQ ID NO: 9)5′-GTAAAACGACGGCCAGT ATTTAGGTGACACTATAG ACACTGACGACATGGTTCTACA-3′NG2SANG-R: M13R-LucF-AdB (M13R = 16; Entire oligo = 58) (SEQ ID NO: 10)5′-AACAGCTATGACCATG CAGTCAAGTAACAACCGCGA TACGGTAGCAGAGACTTGGTCT-3′

One caveat with simultaneous nested PCR is that shorter products aregenerally favored in a multiplex PCR reaction. Therefore, the nestedproduct is likely to be synthesized in far excess of the full lengthproduct containing sequencing sites. However, by altering thethermocycling conditions to favor the full length product, we were ableto significantly increase the yield. Since the gene specific sites allcontain melting temperatures (Tm) around 60° C. and the adapter siteshave Tm closer to 70° C., we ran the first 10 cycles with an annealingtemperature of 55° C. followed by 30 cycles at 62° C. (FIG. 2). Theincreased annealing temperature in the last 30 cycles favors the bindingof the adapter-specific (outer) primers since the gene-specific (inner)primers are much less likely to bind at this elevated temperature.

These thermocycling conditions were compared to standard thermocycling(Thermocycling condition #1) as well as thermocycling indicated forAccessArray Barcode addition (Thermocycling condition #2) (FIG. 3).Various Inner:Outer (P:N; P, PIK3CA-specific inner primers; N, NGSuniversal outer primers) primer ratios were also evaluated for optimalgeneration of full length product (1:10, 2.5:1, 1:4). Results of theseexperiments indicate that our modified thermocycling conditions and anInner:Outer primer ratio of 2.5:1 yielded the maximum full lengthproduct (FIG. 3).

The resulting PCR product was purified from an agarose gel, selectingfor the full length product. BigDye Sequencing was performed using M13forward and reverse sequencing primers. The resulting forward (FIG. 5A)and reverse (FIG. 5A) sequences yielded clean, interpretable resultscovering the entire region of interest.

This design allows for the same sequencing primers to be added to anyamplicon primer set containing adapters A and B to allow for rapidvalidation of any observed mutation in the library. Notably, FIG. 4shows the side-by-side result from our final conditions as compared tothe Fluidigm recommended conditions used for addition of Barcodes in theAccessArray platform. Since the adapter sequences are identical, it isexpected that the efficiency of the reactions would be similar. Indeed,the present method resulted in much higher yield of the full lengthproduct (FIG. 4).

Other Embodiments

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A composition comprising a first oligonucleotidethat comprises, in 5′ to 3′ order, (a) a first region suitable for useas a Sanger sequencing primer; (b) a spacer that is between about 5 andabout 15 nucleotides long; and (c) a third region suitable for use as anext generation sequencing (NGS) primer, and a second oligonucleotidethat comprises, in 5′ to 3′ order, (d) a first region that issubstantially identical to the third region of the firstoligonucleotide; and (e) a second region that is suitable for use as apolymerase chain reaction (PCR) primer, wherein the third region of thefirst oligonucleotide and the first region of the second oligonucleotidehave a melting temperature (T_(m)) that is at least about 5° C. higherthan the T_(m) of the second region of the second oligonucleotide, andwherein the total length of the spacer and the first and second regionsof the second oligonucleotide is at least about 45 nucleotides (nt)long.
 2. The composition of claim 1, wherein the first oligonucleotideis not longer than about 100 nt.
 3. The composition of claim 1, whereinthe second oligonucleotide is not longer than about 60 nt.
 4. Thecomposition of claim 1, wherein the T_(m) of the third region of thefirst oligonucleotide and the first region of the second oligonucleotideis between about 65° C. and about 75° C.
 5. The composition of claim 1,wherein the T_(m) of the second region of the second oligonucleotide isbetween about 55° C. and about 65° C.
 6. The composition of claim 1,wherein the first region of the first oligonucleotide is selected fromTable
 1. 7. The composition of claim 1, wherein the third region of thefirst oligonucleotide and the first region of the second oligonucleotideare selected from Table
 2. 8. The composition of claim 1, furthercomprising a third oligonucleotide that comprises, in 5′ to 3′ order,(a) a first region suitable for use as a Sanger sequencing primer; (b) aspacer that is between about 5 and about 15 nucleotides long; and (c) athird region suitable for use as a next generation sequencing (NGS)primer, and a fourth oligonucleotide that comprises, in 5′ to 3′ order,(d) a first region that is substantially identical to the third regionof the third oligonucleotide; and (e) a second region that is suitablefor use as a polymerase chain reaction (PCR) primer, wherein the thirdregion of the third oligonucleotide and the first region of the fourtholigonucleotide have a melting temperature (T_(m)) that is at leastabout 5° C. higher than the T_(m) of the second region of the fourtholigonucleotide, wherein the total length of the spacer of the thirdoligonucleotide and the first and second regions of the fourtholigonucleotide is at least about 45 nucleotides (nt) long, and whereinthe first region of the first oligonucleotide is substantially differentfrom the first region of the third oligonucleotide and the first regionof the second oligonucleotide is substantially different from the firstregion of the fourth oligonucleotide.
 9. The composition of claim 8,wherein the second region of the second oligonucleotide and the secondregion of the fourth oligonucleotide are suitable for amplifying a humangenomic sequence.
 10. An oligonucleotide comprising, in 5′ to 3′ order:(a) a first region selected from the nucleic acid sequences of Table 1;(b) a spacer that is between about 5 and about 15 nucleotides long; and(c) a third region selected from the nucleic acid sequences of Table 2.11. The oligonucleotide of claim 10, comprising a sequence ofGTAAAACGACGGCCAGTATTTAGGTGACACTATAGACACTGACGACATGGTTCT ACA (SEQ ID NO:9) or AACAGCTATGACCATGCAGTCAAGTAACAACCGCGATACGGTAGCAGAGACTTG GTCT (SEQID NO: 10).
 12. A method for amplifying a nucleotide sequence,comprising: (i) incubating a target nucleotide template with a first andthird oligonucleotides each comprising, in 5′ to 3′ order, (a) a firstregion suitable for use as a Sanger sequencing primer; (b) a spacer thatis between about 5 and about 15 nucleotides long; and (c) a third regionsuitable for use as a next generation sequencing (NGS) primer, and asecond and fourth oligonucleotides comprising, in 5′ to 3′ order, (d)first regions that are substantially identical to the third region ofthe first or third oligonucleotide, respectively; and (e) second regionswhich, in combination, suitable for amplifying the target nucleotidetemplate as a pair of polymerase chain reaction (PCR) primers, whereinthe third regions of the first and third oligonucleotides and the firstregions of the second and fourth oligonucleotides have a meltingtemperature (T_(m)) that is at least about 5° C. higher than the T_(m)of the second regions of the second and fourth oligonucleotide, andwherein the total length of the spacer of the first oligonucleotide andthe first and second regions of the second oligonucleotide is at leastabout 45 nucleotides (nt) long and the total length of the spacer of thethird oligonucleotide and the first and second regions of the fourtholigonucleotide is at least about 45 nucleotides (nt) long, and whereinthe first region of the first oligonucleotide is substantially differentfrom the first region of the third oligonucleotide and the first regionof the second oligonucleotide is substantially different from the firstregion of the fourth oligonucleotide; (ii) performing a plurality of PCRcycles with a first annealing temperature (T_(a)) suitable foramplification using the second regions of the second and fourtholigonucleotides as primers; and (iii) performing a plurality of PCRcycles with a second annealing temperature (T_(a)) suitable foramplification using the third regions of the first and thirdoligonucleotides as primers.
 13. The method of claim 12, wherein thefirst regions of the first and third oligonucleotides are selected fromTable
 1. 14. The method of claim 12, wherein the first regions of thethird and fourth oligonucleotides are selected from Table
 2. 15. Themethod of claim 12, wherein the first Ta is between about 53° C. andabout 57° C.
 16. The method of claim 12, wherein the second Ta isbetween about 59° C. and about 63° C.
 17. The method of claim 12,wherein the ratios of concentrations of the first oligonucleotide to thesecond oligonucleotide and the third oligonucleotide to the fourtholigonucleotide are less than about 1:1.
 18. A composition or kit,comprising a first and second oligonucleotides each comprising, in 5′ to3′ order, (a) a first region suitable for use as a Sanger sequencingprimer; (b) a spacer that is between about 5 and about 15 nucleotideslong; and (c) a third region suitable for use as a next generationsequencing (NGS) primer, wherein the first regions of the first andsecond oligonucleotides are substantially different and the thirdregions of the first and second oligonucleotides are substantiallydifferent.
 19. The composition or kit of claim 18, wherein the firstregions of the first and second oligonucleotides are selected fromTable
 1. 20. The composition or kit of claim 18, wherein the thirdregions of the first and second oligonucleotides are selected from Table2.